CN111979302A - Fluorescent biosensor for detecting transcription factor and detection method and application thereof - Google Patents
Fluorescent biosensor for detecting transcription factor and detection method and application thereof Download PDFInfo
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Abstract
The invention provides a fluorescent biosensor for detecting transcription factors, and a detection method and application thereof, and belongs to the technical field of detection and analysis. The invention provides an ultrasensitive fluorescence strategy based on a bidirectional EXPAR and endonuclease IV auxiliary cycle digestion signal probe to detect transcription factors, and compared with the conventional EXPAR, the bidirectional EXPAR can trigger extension reactions in two directions to generate abundant trigger signals, so that the amplification efficiency is improved. The method is simple to operate, economical, high in sensitivity, low in background and good in specificity, and can be used for realizing ultra-sensitive detection of activity of the transcription factor in the cell, so that the method has good practical application value.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a fluorescence biosensor for detecting transcription factors, and a detection method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Transcription factors can regulate the expression of genes by specifically binding to promoters/enhancers in the DNA duplex. Under the precise control of transcription factors, many normal biological processes, such as cell cycle progression, intracellular maintenance of metabolic and physiological homeostasis, cell differentiation and development, can be performed in order. However, abnormal transcription factor expression levels may lead to a range of diseases, for example, excessive and sustained activation of transcription factor nuclear factors is closely associated with diseases such as cancer, autoimmune diseases, and viral infections. The expression level of transcription factors is not only an important indicator of the disease progression stage, but also a potential diagnostic marker and therapeutic target. Therefore, the sensitive and accurate detection of the activity of the transcription factor is of great significance for studying the pathogenesis of diseases, clinical diagnosis and drug development.
In the current experiments for detecting the activity of the transcription factor, most of the traditional gel electrophoresis and autoradiography detection methods need complex nucleic acid labeling and gel electrophoresis procedures, and the sensitivity is low and time and labor are wasted. The inventors discovered that although the methods can detect the activity of the transcription factor and screen its inhibitors to a certain extent, the methods usually require a long time, require strict temperature control and a large amount of high-concentration proteins and probes, and the detection sensitivity is still to be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fluorescence biosensor for detecting transcription factors and a detection method and application thereof. The invention provides an ultrasensitive fluorescence strategy for detecting transcription factors based on bidirectional EXPAR and endonuclease IV auxiliary circulating digestion signal probes. Compared to conventional EXPAR, bidirectional EXPAR can trigger extension reactions in both directions to generate abundant trigger signals, thereby improving amplification efficiency. The method is simple to operate, economical, high in sensitivity, low in background and good in specificity, and can be used for realizing ultra-sensitive detection of activity of the transcription factor in the cell, so that the method has good practical application value.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a fluorescent biosensor for detecting a transcription factor, the fluorescent biosensor comprising at least a transcription factor binding probe and a signaling probe;
wherein the transcription factor binding probe is formed by hybridizing a p50-s probe and a p50-anti probe; the phosphodiester bonds at the 3' ends of the p50-s probe and the p50-anti probe are both modified by sulfo;
the transcription factor binding probe comprises at least one specific transcription factor recognition element;
the 5' overhang of the transcription factor binding probe comprises the same sequence, and thus can serve as a template for isothermal exponential amplification reaction (EXPAR);
the EXPAR templates each comprise two repetitive sequences and a recognition site for Nb.
The signal probe comprises a purine/pyrimidine (AP) locus, and two ends of a signal probe sequence are provided with a fluorescent group and a quenching group.
In one embodiment of the invention, the signaling probes are labeled with BHQ1 and FAM at the 3 'and 5' ends, respectively.
The fluorescent biosensor also includes Exo III, Klenow fragment polymerase, nt.
When the transcription factor is NF- κ B p50,
the base sequence of the p50-s probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACC C G A GTT TGG GAC TTT CCG TGC (SEQ ID No. 1);
the base sequence of the p50-anti probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACG GAA AGT CCC AAA CT C GG (SEQ ID NO. 2);
wherein represents a thio modification;
the base sequence of the signal probe is as follows: FAM-TCG CAT CXA AGG CAC-BHQ (SEQ ID NO. 3).
In a second aspect of the invention, there is provided a use of a fluorescent biosensor for detecting a transcription factor.
In a third aspect of the present invention, there is provided a method for detecting a transcription factor, the method comprising:
1) adding a sample to be detected into a transcription factor binding probe, adding Exo III for incubation and inactivating enzyme at high temperature;
2) adding the Exo III digestion product into the reaction solution for amplification reaction;
3) and (3) carrying out fluorescence spectrum detection on the solution after the amplification reaction.
In a fourth aspect of the present invention, the fluorescent biosensor and/or the detection method described above is provided for use in detecting transcription factor activity and/or screening transcription factor-related drugs.
Wherein the transcription factor related drug comprises a transcription factor inhibitor and a transcription factor activator.
Although the present invention provides a fluorescence biosensor and a detection method for detecting NF- κ B p50, it should be understood that, based on the concept of the present invention, the substitution of the transcription factor recognition element in the transcription factor binding probe for detecting other transcription factors and related proteins is also conceivable, and thus the present invention is also within the scope of the present invention.
The beneficial technical effects of one or more technical schemes are as follows:
1. the technical scheme can carry out amplification reaction without additionally designing primers and templates, thereby greatly reducing the experiment cost and the experiment complexity.
2. According to the technical scheme, through reasonable phosphorothioate modification of the probe, digestion of the free detection probe by exonuclease can be prevented, so that a fluorescent signal is enhanced.
3. According to the technical scheme, the length of the signal probe is reasonably designed, so that the detected background signal is greatly reduced. And does not need any external separation and elution steps, thus reducing the complexity of the experiment.
4. The specific probe designed by the technical scheme reduces the cost and the complexity of operation and saves resources.
5. The probes combined with the transcription factors and not combined with the transcription factors in the technical scheme participate in the reaction, so that the amplification signals are greatly enhanced, and the resource utilization rate is improved, thereby having good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the detection of transcription factors based on the bidirectional isothermal amplification method in the embodiment of the present invention.
FIG. 2 is a graph of the NF-. kappa. B p 50-initiated EXPAR amplification product analysis in the examples of the present invention, wherein A is a PAGE analysis, and lane M: DNA marker; lane 1: purified recombinant NF-. kappa. B p50+ TF binding probe; lane 2: only TF binding probes are present. B is the normalized fluorescence emission spectra of the reaction product in the absence (control) and in the presence of purified NF- κ B p 50. C is a real-time detection map of the reaction product in the absence (control) and in the purified presence of NF-. kappa. B p50 NF-. kappa. B p 50. Concentration of purified NF-. kappa. B p50 was 8X 10-7Molar, TF binding probe concentration of 500 nanomolar.
FIG. 3A is a graph showing fluorescence spectra of NF- κ B p50 at various concentrations in examples of the invention;
FIG. 3B shows fluorescence intensity at 521nm and NF- κ B p50 concentration at 6.4 × 10 in example of the present invention-14Molar to 8X 10-7Linear relationship between the logarithms in the molar range. The concentration of TF binding probe was 500 nanomolar. Error bars show the standard deviation of three independent experiments.
FIG. 4 shows a representation of the presence of an embodiment of the present invention500nM TF binding probe (control), 500nM TF binding probe +1 mg/ml BSA, 500nM TF binding probe + 8X 10-7Mol c-Jun, 500nM TF binding probes + 8X 10-7Molar p65, 500 nanomolar TF binding probes + 8X 10-7Fluorescence intensity measurement at molar NF-. kappa. B p 50. Error bars show the standard deviation of three independent experiments.
FIG. 5A is a fluorescence emission spectrum measured in the absence and presence of an inhibitor in an example of the present invention.
FIG. 5B shows fluorescence intensity measured in the absence and presence of inhibitors in examples of the invention. 500 nmol of TF binding probe, 0.25 mg/ml of nuclear extract and 20 micromol of rubescensin were used in the experiment. Error bars show the standard deviation of three independent experiments.
FIG. 6A is a fluorescence emission spectrum in response to different concentrations of nuclear extracts in examples of the present invention.
FIG. 6B is a graph showing 4X 10 log of fluorescence intensity at 521nm versus nuclear extract concentration in examples of the present invention-5To 2.5X 10-1Linear relationship between mg/ml. The concentration of TF binding probe was 500 nanomolar. Error bars show the standard deviation of three independent experiments.
FIG. 7A is a graph showing the F/F signal probe pairs at different concentrations at a fixed Endo IV (5 units) in an example of the present invention0The effect of the value;
FIG. 7B is a graph showing the response of different amounts of Endo IV induced F/F at a fixed concentration of signaling probe (300 nanomolar) in accordance with an example of the present invention0A change in value;
FIG. 7C is a graph showing the change of fluorescence intensity with reaction time in examples of the present invention. The concentration of pure NF-kappa B p50 was 8X 10-7Molar, TF binding probe concentration of 500 nanomolar. Error bars show the standard deviation of three independent experiments.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, in the current experiments for detecting the activity of the transcription factor, most of the traditional gel electrophoresis and autoradiography detection methods require complex nucleic acid labeling and gel electrophoresis procedures, and the sensitivity is low and time and labor are wasted. The later development of new technologies for detecting activity of transcription factors, including labeling signal probes with double fluorescent dyes and designing double stem-loop molecular beacons for auxiliary detection, and various recent and emerging amplification strategies, such as real-time Polymerase Chain Reaction (PCR), isothermal exponential amplification reaction (EXPAR), helicase dependent amplification (HAD), Rolling Circle Amplification (RCA), etc., requires special design of primers and/or templates, and although these methods can detect activity of transcription factors and screen inhibitors thereof to a certain extent, these methods often require long time consumption, require strict temperature control and large amount of high-concentration proteins and probes, and detection sensitivity still needs to be improved.
In view of the above, the present invention provides an ultrasensitive fluorescence strategy based on bidirectional EXPAR and endonuclease IV assisted cycle digestion signal probes to detect transcription factors. Compared to conventional EXPAR, bidirectional EXPAR can trigger extension reactions in both directions to generate abundant trigger signals, thereby improving amplification efficiency. The method is simple to operate, economical, high in sensitivity, low in background and good in specificity, and can be used for realizing ultra-sensitive detection of transcription factor activity in cells.
Specifically, in one exemplary embodiment of the present invention, a fluorescent biosensor for detecting a transcription factor is provided, the fluorescent biosensor including at least a transcription factor binding probe and a signaling probe;
wherein the transcription factor binding probe is formed by hybridizing a p50-s probe and a p50-anti probe; the phosphodiester bonds at the 3' ends of the p50-s probe and the p50-anti probe are both modified by sulfo;
the transcription factor binding probe comprises at least one specific transcription factor recognition element;
the 5' overhang of the transcription factor binding probe comprises the same sequence, and thus can serve as a template for isothermal exponential amplification reaction (EXPAR);
the EXPAR templates each comprise two repetitive sequences and a recognition site for Nb.
The signal probe comprises a purine/pyrimidine (AP) locus, and two ends of a signal probe sequence are provided with a fluorescent group and a quenching group.
In one or more embodiments of the invention, the signaling probes are labeled with BHQ1 and FAM at the 3 'and 5' ends, respectively.
In one or more embodiments of the invention, the fluorescent biosensor further comprises Exo III, Klenow fragment polymerase, nt.
In one or more embodiments of the invention, when the transcription factor is NF- κ B p50,
the base sequence of the p50-s probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACC C G A GTT TGG GAC TTT CCG TGC
(SEQ ID NO.1)；
The base sequence of the p50-anti probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACG GAA AGT CCC AAA CT C GG (SEQ ID NO. 2);
wherein represents a thio modification;
the base sequence of the signal probe is as follows: FAM-TCG CAT CXA AGG CAC-BHQ (SEQ ID NO. 3).
In one or more embodiments of the present invention, there is provided a use of a fluorescent biosensor for detecting a transcription factor. Such transcription factors include, but are not limited to NF-. kappa. B p 50.
In one or more embodiments of the present invention, there is provided a method of detecting a transcription factor, the method comprising:
1) adding a sample to be detected into a transcription factor binding probe, adding Exo III for incubation and inactivating enzyme at high temperature;
2) adding the Exo III digestion product into the reaction solution for amplification reaction;
3) and (3) carrying out fluorescence spectrum detection on the solution after the amplification reaction.
In the step S1, the incubation reaction condition is 35-40 ℃ (preferably 37 ℃), and the reaction time is 5-20 min (preferably 10 min);
the high-temperature enzyme deactivation reaction condition is 70-90 ℃ (preferably 80 ℃), and the reaction condition is 5-20 minutes (preferably 10 minutes);
in the step S2, the reaction solution at least includes dNTP, Klenow fragment polymerase, nt.bsti, signaling probe, and Endo IV.
Wherein the concentration of the signal probe is 100-500 nm, and preferably 300 nm; the using amount of the Endo IV is 1-9 units, and preferably 5 units.
The amplification reaction conditions are specifically as follows: 35-40 ℃ (preferably 37 ℃), and the reaction time is 5-100 min (preferably 60 min);
the step S3) includes the following specific steps: the fluorescence spectrum was detected with a fluorescence spectrophotometer and data analysis was performed at an excitation wavelength of 490nm and fluorescence intensity at 521 nm.
In one or more embodiments of the present invention, the fluorescent biosensor and/or the detection method described above is provided for use in detecting transcription factor activity and/or screening transcription factor-related drugs.
Wherein the transcription factor related drug comprises a transcription factor inhibitor and a transcription factor activator.
Although the present invention provides a fluorescence biosensor and a detection method for detecting NF- κ B p50, it should be understood that, based on the concept of the present invention, the substitution of the transcription factor recognition element in the transcription factor binding probe for detecting other transcription factors and related proteins is also conceivable, and thus the present invention is also within the scope of the present invention.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The base sequence of p50-s is: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACC C G A GTT TGG GAC TTT CCG TGC (SEQ ID No. 1); the base sequence of p50-anti is: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACG GAA AGT CCC AAA CT C GG (SEQ ID NO. 2); wherein represents a thio modification; the base sequence of the signal probe is as follows: FAM-TCG CAT CXA AGG CAC-BHQ (SEQ ID NO. 3).
Examples
protein-DNA interaction and exonuclease digestion: 10 micromolar p50-s and 10 micromolar p50-anti were incubated in annealing buffer containing 100 mM NaCl and 10 mM Tris-HCl (pH 7.5) at 95 ℃ for 5 minutes, then slowly cooled to room temperature. Various concentrations of 1. mu.l of purified recombinant NF-. kappa. B p50 and 500nM TF binding probe were added to 10. mu.l of binding buffer (10 mM Tris-HCl (pH 7.5), 100 mM KCl, 2 mM MgCl20.1 mmol EDTA, 0.1 mg/ml yeast tRNA, 10% glycerol, 0.25 mmol DTT). Then 10 units of Exo III and 1. mu.l of 10 XNEB buffer 1 were added, incubated at 37 ℃ for a further 10 minutes and then heated at 80 ℃ for 10 minutes to inactivate the Exo III.
Amplification reaction and fluorescence measurement: the Exo III digested product was added to 50 μ l of a reaction solution containing 500 nmol dNTP, 2.5 units Klenow fragment polymerase, 5 units nt.bsti, 300 nmol signal probe, 5 units Endo IV, 5 μ l 10 × NEBuffer 2, and 5 μ l 10 × CutSmart, followed by incubation at 37 ℃ for 60 minutes. The fluorescence spectrum was measured at an excitation wavelength of 490nm using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan), and the fluorescence intensity at 521nm was recorded for data analysis.
Gel electrophoresis analysis: the Exo III digested product was added to 50. mu.l of a reaction solution containing 500 nmol of dNTP, 2.5 units of Klenow Fragment polymerase, 5 units of Nt.BstI, 5. mu.l of 10 XNEBuffer 2 and 5. mu.l of 10 XCutSmart, followed by incubation at 37 ℃ for 60 minutes. The products of the bipartite EXPAR were mixed with the fluorescent indicator SYBR Gold and the mixture was separated in 1 XTTris-borate EDTA (TBE) buffer (89 mM Tris-HCl, 1 XTTris-borate EDTA (TBE)) by electrophoresis on a 12% native polyacrylamide gel (PAGE). The solution was left at room temperature for 45 minutes at constant pressure of 110V in (89 mmol of boric acid, 2 mmol of EDTA, pH 8.3). Images of gel electrophoresis were visualized by the ChemiDoc MP Imaging system (helecklesch, california, usa).
Inhibitor experiments: for the determination of transcription factor inhibition, 20 micromolar rubescensin was incubated with 0.25 mg/ml nuclear extract and 500 nanomolar TF binding probes in 10 microliters of binding buffer at 37 ℃ for 30 minutes. The subsequent reaction followed the procedure described above and the fluorescence intensity was measured as described above.
Cell culture and preparation of cell extracts: hela cells (human cervical cancer cell line) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin and placed in an incubator containing 5% carbon dioxide at 37 ℃. After incubating HeLa cells with 20 ng/ml TNF- α (Invitrogen, USA) for 40 minutes, nuclear extracts were prepared using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. The concentration of total protein was measured using a Bradford based assay.
The principle of the transcription factor activity assay in this example is depicted in FIG. 1. The p50-s probe can hybridize to the p50-anti probe to form a TF binding probe that contains a specific TF recognition element and the 5' overhang of the binding probe contains the same sequence as the template for the EXPAR. Each EXPAR template contains two repeats and a recognition site for nb. The detection scheme comprises three steps: (1) TF-DNA interaction and exonuclease digestion, (2) TF-DNA interaction induced bidirectional EXPAR, and (3) circular digestion of the Endo IV drive signal probe to generate a fluorescent signal. In this assay, NF-. kappa. B p50 specifically binds to the protein binding site in the TF binding probe, blocking the digestion of the probe by exonuclease III (Exo III) while leaving the TF binding probe in a double-stranded configuration. The TF binding probe can serve as both a template and a primer to initiate polymerization reactions from both directions in the presence of KF DNA polymerase, generating duplex DNA, each duplex containing specific recognition sites for the two enzymes nb. Btsi enzymes then cleave specific recognition sites in dsDNA, thereby generating abundant trigger signals. Notably, the presence of NF- κ B p50 prevented digestion of TF-binding probes by Exo III, the excess p50-s probe of TF-binding probes was digested from the 3 'terminal portion of the double-stranded DNA by Exo III up to the position of phosphorothioate modification (fig. 1, asterisk), while the excess p50-anti probe of TF-binding probes was not digested by Exo III, due to steric hindrance at the 3' end of p50-anti probe by phosphorothioate modification hindering enzymatic digestion. The resulting triggers can bind to the EXPAR template regions of the p50-s probe and the p50-anti probe, induce a cycling reaction of polymerization, digestion and displacement, and ultimately lead to exponential amplification to produce large numbers of triggers. The signaling probes were labeled with BHQ1 and FAM at the 3 'and 5' ends, respectively, and modified with a purine/pyrimidine (AP) site in the middle of the sequence. The resulting trigger probe can hybridize to the signaling probe to form DNA duplexes, each duplex containing an intact AP site, which can be cleaved by Endo IV to generate a unique fluorescent signal and release the trigger simultaneously. The released trigger can further bind to a new signaling probe to initiate cyclic cleavage of the signaling probe, thereby significantly enhancing the fluorescent signal. It should be noted that not only the TF-binding probe bound to p50, but also the binding of free TF-binding probe to the trigger was involved in the NF-. kappa. B p 50-triggered EXPAR amplification (FIG. 1). Therefore, in a reaction system, whether the probe is combined with TF or not, all TF-combined probes participate in the amplification reaction, and the amplification efficiency is greatly improved. In contrast, in the absence of TF, free TF-bound probe was digested by Exo III, so that no bidirectional EXPAR occurred. As a result, no trigger was generated, and no fluorescent signal was observed.
1. Experimental verification of principle
To investigate whether the presence of TF could prevent the probe from being digested by Exo III and initiate bidirectional EXPAR, non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to analyze the reaction products. As shown in FIG. 2, in the absence of NF-. kappa. B p50, the p50-s probe was digested by Exo III from the 3 'terminal portion of the double-stranded DNA up to the site of phosphorothioate modification, whereas the p50-anti probe was not digested due to phosphorothioate modification at the 3' end. Thus, in the presence of only TF-binding probe, only the 41nt band generated by the partially digested p50-s probe, and the p50-anti probe (55nt) were observed (FIG. 2A, lane 2). In the presence of NF-. kappa. B p50, the interaction of TF-binding probes with p50 prevented Exo III digestion, free TF-binding probes were partially digested by Exo III, and the remaining TF-binding probes triggered bidirectional EXPAR to generate large amounts of triggers. Thus, amplified bands (94bp, 55bp and 41bp) and a trigger band (21nt) were observed in the presence of the TF binding probe and NF- κ B p50 (FIG. 2A, lane 1). To further verify the feasibility of the proposed assay, the fluorescence signal was monitored under different experimental conditions. In the presence of NF-. kappa. B p50, a strong fluorescence signal was observed, which was characterized by an emission peak at 521nm (FIG. 2B). However, no significant fluorescence signal was observed in the control group without the addition of NF- κ B p50 (FIG. 2B). In addition, real-time fluorescence measurements were used to validate the method. In the presence of NF- κ B p50, it induced an enhanced fluorescence signal within the first 20 minutes and then reached a plateau (FIG. 2C). In contrast, no fluorescence signal was observed in the control group without NF- κ B p50 (FIG. 2C). These results indicate that TF can induce bidirectional EXPAR and initiate subsequent Endo IV driven signal probe cycle digestion to generate enhanced fluorescent signals.
2. Sensitivity test
To investigate the detection sensitivity of this method, the change in fluorescence intensity at different concentrations of NF- κ B p50 was measured. As shown in FIG. 3A, as the NF- κ B p50 concentration increased from 0 to 8 × 10-7Molar, fluorescence intensity increased at 6.4X 10-14Molar to 8X 10-7There was a linear correlation between fluorescence intensity and the logarithm of the concentration of NF- κ B p50 over a dynamic range of 7 orders of magnitude in moles (FIG. 3B). The regression equation is that F is 6192.62+437.43log10C(R20.987), where F is the fluorescence intensity and C is the concentration of NF- κ B p 50. The detection limit calculated from the mean signal of the blank plus 3 times the standard deviation is 1.29X 10-14And (3) mol. With Fluorescence Resonance Energy Transfer (FRET) analysis based on molecular beacons (2X 10)-8Mole), the sensitivity of the method is improved by 6 orders of magnitude, and the method is compared with a DNA-Ag nano cluster molecular beacon (AgMBs) based fluorescence experiment (1 multiplied by 10)-11Molarity) and electrochemical assays based on DNase (8X 10)-11Mole) experiment, the sensitivity is improved by 3 orders of magnitude, and the method is more than colorimetric analysis (3.8 multiplied by 10) based on isothermal exponential amplification-12Mole) by 2 orders of magnitude higher, with helicase dependent amplification-based methods (9.3X 10)-13Molar) compared to 1 order of magnitude. A significant advantage of this method over the reported TF assay method is that both bound and unbound TF-bound probes participate in the amplification reaction as long as NF- κ B p50 is present. The increase in sensitivity of the proposed method can be attributed to three factors: (1) the TF-DNA interaction induces high amplification efficiency of the bidirectional EXPAR. (2) Both bound and unbound TF-binding probes participate in the amplification reaction; (3) the Endo IV-driven signal probe cycle digestion produces an enhanced fluorescent signal.
3. Detection specificity
The specificity of the proposed method depends mainly on the specific binding interaction between TF and TF binding probes. To evaluate the specificity of the assay, Bovine Serum Albumin (BSA), cellular Jun (c-Jun), NF- κ B p65(p65) were selected as model interferents. BSA is an unrelated protein. c-Jun is a member of the (bZIP) family of dimeric transcriptional activators that can recognize and bind to the enhancer heptameric motif 5 '-TGACGTCA-3'. Like NF-. kappa. B p50, NF-. kappa. B p65 belongs to the NF-. kappa.B family, but has a different sequence recognition site from p 50. As shown in FIG. 4, a significant fluorescent signal was observed in the presence of NF- κ B p50, whereas no significant fluorescent signal was observed in the presence of BSA, c-Jun protein and p 65. Although NF-. kappa. B p65 is a member of the NF-. kappa.B family, no significant fluorescent signal was obtained due to the difference in the binding site between NF-. kappa. B p65 and NF-. kappa. B p 50. Only NF-. kappa. B p50 was able to bind efficiently to specific TF-binding probes to initiate bidirectional EXPAR amplification. These results demonstrate the good specificity of the proposed method for NF-. kappa. B p 50.
4. Inhibitor detection
NF-. kappa.B exists in the cytoplasm in an inactive state and is activated by various stimulators including proinflammatory cytokines, oxidative stress and hyperglycemia. NF- κ B may regulate the inducible expression of genes associated with tumor promotion, angiogenesis and metastasis. Therefore, NF- κ B has become a major target for drug development and cancer therapy. To investigate the ability of the present method to assay NF- κ B p50 inhibition, oridonin was used as a model NF- κ B inhibitor. Rubescensin directly interferes with the DNA binding activity of NF- κ B on its responsive DNA sequence, and fig. 5A shows the change in intensity of the fluorescence spectrum in the presence and absence of inhibitor. A significant decrease in fluorescence intensity was observed in the presence of rubescensin (FIG. 5B), indicating that rubescensin can inhibit the binding of NF- κ B p50 to TF binding probes. These results indicate that the proposed method can be used to screen for TF inhibitors.
5. Detection of endogenous TF
Tumor necrosis factor-a (TNF-a) stimulates NF- κ B p50 activity inside HeLa cells. To demonstrate the ability of the proposed method to perform an actual sample analysis, the activity of endogenous NF-. kappa. B p50 was measured in crude HeLa nuclei by the induction of TNF-. alpha.. As shown in FIG. 6A, the fluorescence intensity varied from 0 to 2.5X 10 with the concentration of the nuclear extract-1Mg/ml increase but monotonically. In addition, fluorescence intensity and nuclear intensityThe concentration of the extract is 4 × 10-5To 2.5X 10-1The logarithm in the mg/ml range is linearly related (fig. 6B), and the regression equation is that F is 3301.06+675.75log10 C(R20.995), where F is the fluorescence intensity and C is the concentration of the nuclear extract. The detection limit calculated by evaluating the average response of the control group plus three times the standard deviation was 2.8X 10-5Mg/ml. And a near-infrared fluorescence solid-phase rolling circle amplification (NIRF-sRCA) -based method (6.25X 10)-2Mg/ml) and the reported FRET method (5X 10)-2Mg/ml) compared to 3 orders of magnitude improvement in sensitivity. These results clearly indicate that this method can be used for sensitive detection of endogenous NF-. kappa. B p50 activity.
6. Optimization of conditions
To obtain better analytical performance, three experimental parameters were optimized, including the concentration of signaling probe, the amount of Endo IV used and the time of reaction. As shown in FIG. 7A, the F/F0 value gradually increased as the concentration of signaling probe increased from 100 nanomolar to 300 nanomolar, and then the signal gradually decreased at concentrations above 300 nanomolar (F and F)0Fluorescence intensity in the presence and absence of p50 protein, respectively). Therefore, the optimal concentration of signaling probe is 300 nanomolar. The effect of the amount of Endo IV was further investigated at a fixed signal probe concentration (300 nanomolar). F/F as shown in FIG. 7B0The values increased with increasing Endo IV and reached a plateau at 5 units (F and F)0Fluorescence intensity in the presence and absence of p50 protein, respectively). Thus, 5 units of Endo IV were used in subsequent studies. The reaction time was also optimized, as shown in fig. 7C, the fluorescence intensity increased with the reaction time from 0 to 60 minutes and reached a plateau at 60 minutes. Therefore, 60 minutes was used as the optimal reaction time in the subsequent studies.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
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Claims (10)
1. A fluorescent biosensor for detecting a transcription factor, comprising at least a transcription factor binding probe and a signaling probe;
wherein the transcription factor binding probe is formed by hybridizing a p50-s probe and a p50-anti probe; the phosphodiester bonds at the 3' ends of the p50-s probe and the p50-anti probe are both modified by sulfo;
the transcription factor binding probe comprises at least one specific transcription factor recognition element;
the 5' overhang of the transcription factor binding probe comprises the same sequence as the EXPAR template;
the EXPAR templates comprise two repetitive sequences and recognition sites of Nb.BtsI, and the recognition sites take transcription factor recognition elements as primers to start an extension reaction;
the signal probe comprises a purine/pyrimidine (AP) locus, and two ends of a signal probe sequence are provided with a fluorescent group and a quenching group.
2. The fluorescent biosensor for detecting a transcription factor as claimed in claim 1, wherein the signaling probes are labeled with BHQ1 and FAM at the 3 'and 5' ends, respectively.
3. The fluorescent biosensor for detecting transcription factors as claimed in claim 1, wherein the fluorescent biosensor further comprises Exo III, Klenow fragment polymerase, nt.
4. The fluorescence biosensor for detecting a transcription factor as claimed in claim 1, wherein when the transcription factor is NF- κ B p50,
the base sequence of the p50-s probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACC C G A GTT TGG GAC TTT CCG TGC (SEQ ID No. 1);
the base sequence of the p50-anti probe is as follows: TCG CAT CTA AGG CAC GCA GTG AGT CGC ATC TAA GGC ACG GAA AGT CCC AAA CT C GG (SEQ ID NO. 2);
wherein represents a thio modification;
the base sequence of the signal probe is as follows: FAM-TCG CAT CXA AGG CAC-BHQ (SEQ ID NO. 3).
5. Use of the fluorescent biosensor according to any one of claims 1 to 4 for detecting transcription factors.
6. A method for detecting a transcription factor, comprising detecting with the fluorescent biosensor of any one of claims 1-4:
1) adding a sample to be detected into a transcription factor binding probe, adding Exo III for incubation and inactivating enzyme at high temperature;
2) adding the Exo III digestion product into the reaction solution for amplification reaction;
3) and (3) carrying out fluorescence spectrum detection on the solution after the amplification reaction.
7. The method according to claim 6, wherein in the step S1, the incubation reaction condition is 35-40 ℃ (preferably 37 ℃) and the reaction time is 5-20 min (preferably 10 min);
the high-temperature enzyme deactivation reaction condition is 70-90 ℃ (preferably 80 ℃), and the reaction condition is 5-20 minutes (preferably 10 minutes).
8. The method of claim 6, wherein in step S2, the reaction solution comprises at least dNTP, Klenow fragment polymerase, Nt.BstI, signaling probe, and Endo IV;
wherein the concentration of the signal probe is 100-500 nm, and preferably 300 nm; the using amount of the Endo IV is 1-9 units, preferably 5 units;
the amplification reaction conditions are specifically as follows: 35-40 ℃ (preferably 37 ℃), and the reaction time is 50-70 min (preferably 60 min).
9. The method as claimed in claim 6, wherein the step S3) is specifically performed by: the fluorescence spectrum was detected with a fluorescence spectrophotometer and data analysis was performed at an excitation wavelength of 490nm and fluorescence intensity at 521 nm.
10. Use of the fluorescence biosensor according to any one of claims 1 to 4 and/or the detection method according to any one of claims 6 to 9 for detecting transcription factor activity and/or screening transcription factor-related drugs;
preferably, the transcription factor-related drug includes a transcription factor inhibitor and a transcription factor activator.
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| CN113373200A (en) * | 2021-03-18 | 2021-09-10 | 南京师范大学 | Method for detecting transcription factor NF-kappa B p50 by biosensor |
| CN113373200B (en) * | 2021-03-18 | 2023-07-21 | 南京师范大学 | Method for detecting transcription factor NF-kappa B p50 by biosensor |
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